DESCRIPTION (applicant's description): The long-term objective of this research project is to enhance our understanding of protein chemistry and biology. The proposed research is focused on ribonuclease A (RNase A), a small protein that efficiently catalyzes the cleavage of the P-0 bond of RNA. Classic work on RNase A has contributed much information to protein science. In the proposed research, this wealth of information is combined with modern ideas and methods to achieve six specific aims. The specific aims of this research project are (1) Staudinger ligation-develop a powerful new method for creating semisynthetic proteins; (2) peptide bond isosteres-determine the contribution of a main-chain NH to catalysis; (3) nonnatural modules-determine the contribution to conformational stability of disulfide bond isosteres, a covalent mimic of a hydrogen bond, a metathesis crosslink, and (3) turn mimics; (4) hydroxyurea inhibitor-create a new type of ribonuclease inhibitor; (5) function in vivo-determine the true physiological role of pancreatic ribonuclease and the cytosolic ribonuclease inhibitor protein (which binds to pancreatic ribonuclease with high affinity) by creating and analyzing knockout mice; and (6) three-dimensional structures-determine at atomic resolution the structures of the most informative protein variants created herein. Aims 1 - 3 use RNase A as a model system to test hypotheses of broad applicability. In particular, these aims use and extend new techniques that combine recombinant DNA technology with synthetic organic chemistry to create proteins containing nonnatural functionalities of great potential utility. Aims 4 - 6 address the most meaningful issues on the function and structure of RNase A itself. The proposed experiments use methods and ideas from organic chemistry, biochemistry, biophysics, molecular biology, and molecular genetics to provide new insights into protein chemistry and biology, and could ultimately lead to novel proteins for biomedical analyses and therapies.